The present patent application claims the right of priority under 35 U.S.C. xc2xa7119(a)-(d) of German Patent Application Numbers 199 09 786.0, filed Mar. 5, 1999, and 199 51 734.7, filed Oct. 27, 1999.
This invention relates to esters of oligo- and polysaccharides which are regio-selectively substituted, predominantly at the C2 position of the anhydroglucose unit (AGU), and also relates to a method of producing them using enzymes and/or defined salts as catalysts for ester formation. Regioselectively substituted esters of this type have basic properties which are different from those of conventional, randomly functionalised products.
It is known that when oligo- and polysaccharides are subjected to esterification by conventional methods, products are formed which exhibit a random distribution of the esters groups within the AGU and along the chain. This randomness depends on the accessibility to electrons or on the spatial accessibility of the individual hydroxyl groups. Thus in starch, for example, the hydroxyl group in the C6 position is a primary group which is very sterically accessible and which thus exhibits the highest accessibility during heterogeneous reactions in particular. The hydroxyl function of the C2 position forms the basis of the electronic effect of the adjacent glycoside bond and of the electron-attracting properties of the ring oxygen. In homogeneous processes, it has been shown that the C2 position reacts first for this reason. In none of the aforementioned situations, however, is the complete reaction of only one hydroxyl group achieved.
By utilising differences in accessibilities such as these, hydroxyl groups can be selectively blocked with the aid of protective groups, which are generally bulky and can easily be split off, so that regioselective derivatives are formed in subsequent reactions.
Examples of protective groups such as these include triphenylmethyl groups or bulky organosilicon entities such as t-hexyl- or tert-butyl-dimethylsilyl groups.
However, this type of synthesis has the decisive disadvantage that at least two additional reaction steps are necessary due to the introduction of the protective group and the separation thereof. Other disadvantages are the fact that the separation of the protective groups is sometimes incomplete, which means that the cleavage products which are formed thereby, and which are sometimes toxic, have to be removed without leaving a residue, as well as the breakdown of the polysaccharide chain which is possible under the conditions of cleavage and which changes the properties of the product.
When a plurality of reactive centres, e.g. hydroxyl groups, exists in a molecule, enzymes are capable of catalysing direct, selective esterification reactions. In this connection, each enzyme has a certain folded (native) structure which is essential for its specific biocatalytic activity in the physiological medium concerned. It has been shown in numerous publications, however, that many enzymes are also active in organic solvents, i.e. are active irrespective of their native structure, size and function. Enzymes generally exhibit a high activity in nonpolar solvents, whereas only very low activities are found in relatively polar media (Biotechnol. Bioeng. 30 (1987), 81-87). Enzymes are insoluble in the latter organic solvents.
Numerous enzyme-catalysed reactions of this type have already been carried out on low molecular weight mono- and disaccharides in organic solvents (FEMS Microbiol. Rev. 16 (1995), 193-211; J. Prakt. Chem. 335 (1993), 109-127; Synthesis 1992, 895-910; WO 97/36000; WO 95/23871). In these reactions lipases have primarily been used as the enzymes, although esterases and proteases have also been used, and solvents such as tetrahydrofuran, pyridine and N,N-dimethylformamide have been employed. Enzymatic esterifications can also be effected in an aqueous buffer solution. One disadvantage here is that the acylating reagent, which has a character similar to that of a fatty acid, is insoluble in the aqueous buffer solution and can thus only be suspended therein. The substrate which is to be esterified is present in dissolved form (DE-A-34 30 944,1992; JP-A-63191802).
Glucans generally result in the formation of esters at primary hydroxyl groups. In some cases, esterification also occurs at secondary hydroxyl groups (J. am. Chem. Soc. 109 (1987), 3977-3981; Enzyme Microb. Technol. 20 (1997), 225-228). Compounds which comprise electron-attracting groups are generally used for the aforementioned enzymatic esterifications, such as vinyl esters (Biotechnol. Lett. 19 (1997), 511-514) or trihalogenoethyl esters (Tetrahedron 54 (1998), 3971-3982); esterification by a vinyl ester constitutes an irreversible reaction, since the vinyl alcohol which is formed is removed as acetaldehyde from the reaction equilibrium. Other reactive compounds include carboxylic anhydrides and esters of carbonic acid.
Diesters of dicarboxylic acids have also been used for reaction with mono- and disaccharides. In this manner, it has proved possible to synthesise new saccharide-based copolymers, as disclosed in U.S. Pat. No. 5,270,42 1 and U.S. Pat. No. 5,618,933.
In the publications described above, the substrate and the acylating reagent are generally dissolved in an organic solvent, in which the enzyme is then suspended. Enzymatic esterifications cannot be effected on polysaccharides, particularly glucans, by the methods cited above, since the undissolved or unswollen polymer is not accessible to the enzyme or to enzyme catalysis. Polysaccharides can be esterified enzymatically in a heterogeneous phase, however (WO 96/13632, DE-A-34 30 944). These heterogeneous reactions only proceed at the surface of the polymer particles, due to which inhomogeneously esterifed polysaccharide derivatives are formed, i.e. polysaccharide derivatives which comprise a non-uniform distribution of substituents along the polymer chain. Other disadvantages of this method of esterification are that low yields of product are obtained and products are formed which comprise a low degree of selectivity as regards the type of substitution in the anhydroglucose unit.
Conventional heterogeneous esterifications are often conducted in water as a suspension medium (U.S. Pat. No. 5,703,226, 1997). Homogeneous reactions are conducted in organic solvents or in the acylating reagent directly (U.S. Pat. No. 5,714,601, 1998; WO 96/14342). The corresponding carboxylic anhydrides or vinyl esters are generally used as acylating agents. Esterifications or transesterifications of this type are mostly catalysed by alkalies, wherein suitable catalysts include alkali hydroxides, salts of mineral acids or organic amines.
The object of the present invention was to enable esters of oligo- and polysaccharides to be obtained which are homogeneously and regioselectively substituted at the C2 position of the anhydroglucose unit (AGU). Success has now been achieved according to the invention in producing esters of oligo- and polysaccharides which are homogeneously and regioselectively substituted at the C2 position of the AGU. The present invention therefore relates to oligo- and polysaccharide esters which are esterified regioselectively, with ester groups at the C2 position of the AGU preferably amounting to at least 90%, most preferably 90 to 98%, of the total degree of substitution (partial average degree of substitution AS at the C2 position of the AGU with respect to the total AS). The oligo- and polysaccharide esters which are regioselectively substituted at the C2 position and which are particularly preferred according to the invention are esters of starch or starch derivatives, particularly hydroxyethylstarch or hydroxypropylstarch. The present invention also relates to esters, which are regioselectively substituted at the C2 position, of cellulose, cellulose esters or cellulose ethers, particularly of hydroxyethyl cellulose, methyl cellulose, pullulan or maltose. The oligo- and polysaccharide esters according to the invention are obtainable by reaction with esterification reagents from the group comprising vinyl esters, carboxylic anhydrides and trihalogenoethyl esters, as well as lactones, which are described in more detail below in the description of the method. Oligo- and polysaccharide esters which regioselectively substituted at the C2 position and which are particularly preferred according to the invention are 2-O-propionyl-starch, 2-O-butyrylstarch, 2-O-benzoylstarch, 2-O-laurylstarch, 2-O-methoxy-carbonylstarch, 2-O-acryloylstarch and 2-O-methacryloylstarch, most preferably 2-O-acetylstarch. According to the invention, the oligo- and polysaccharides which are regioselectively substituted at the C2 position can be esterified at the remaining OH groups of the AGU with other ester groups which are not identical to the ester groups at the C2 position.
The present invention further relates to a method of producing the compounds according to the invention in the presence of an organic solvent, which method is catalysed by enzyme or salt and is carried out on a dissolved or highly swollen oligo- or polysaccharide.
Starting from the disadvantages of the known methods which were described above, e.g. low yields of product and low degrees of regioselectivity, it has thus proved possible, using catalysis by means of enzymes, to effect the esterification of oligo- and polysaccharides, which are dissolved in organic solvents or which are strongly swollen, regioselectively at the secondary hydroxyl group of the C2 position, and has also proved possible to effect said esterification in combination with the esterification of the primary hydroxyl group of the C6 position of the AGU. The method results in very high product yields, and the partial degrees of substitution can be varied and adjusted over wide limits. Particularly high regioselectivities are achieved with oligo- and polysaccharides which comprise an xcex1-(1,4)-glycoside linkage, particularly starch. However, the method can also be applied to oligo- and polysaccharides which comprise a xcex2-(1,4)-glycoside linkage. Organic solvents which are suitable in principle are those in which the oligo- and polysaccharides which are used exhibit considerable swelling or dissolve, and in which the enzymes used exhibit satisfactory activity. Polar organic solvents such as Dimethyl sulphoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), pyridine and N-methylmorpholine-N-oxide (NMMO) can therefore be used as solvents, as can mixtures of the aforementioned solvents. DMSO is preferred. The enzymes surprisingly exhibit high activity, particularly in DMSO, and products which are esterified strictly regioselectively are obtained in a single-stage reaction. These esters can be converted into regioselectively substituted mixed derivatives, such as esters and ethers, by subsequent, selective reactions.
The esterification can be carried out on polysaccharides, preferably on starch from various natural sources and with wide range of amylose contents and molecular weights, and on starch derivatives, particularly hydroxyethylstarches or hydroxy-propylstarches, and can also be carried out on cellulose, cellulose derivatives, pullulan, pullulan derivatives and oligosaccharides.
Proteases of all types can be used as enzymes. The preferred proteases are serine-, cysteine-, asparagine- and metalloproteases. The proteases are preferably dissolved in a phosphate buffer, or alternatively in a carbonate buffer, within a pH range of 4-9 depending on the enzyme, preferably at pH 7-8, and are subsequently lyophilised. The preferred proteases are proteinase N and subtilisin of Bacillus subtilis, proteinase 2A of Aspergillus oryzae, proteinase 6 of Aspergillus sp., a-chymotrypsin, papain, renin and thermolysin.
Esters of general formula 
are preferably used as esterification reagents, wherein R2 preferably denotes a saturated or unsaturated alkyl group comprising 2 to 6 C atoms, or an unsaturated or saturated, branched or unbranched trihalogenoalkane radical comprising 2 to 4 C atoms, particularly vinyl, trihalogenoethyl or alkyl. R1 is preferably an alkyl group comprising 2-18 C atoms, which can be saturated, unsaturated, straight-chain, branched or cyclic and which can optionally be substituted, or an aryl group (which is optionally substituted). R1 is most preferably selected from the group comprising acetyl, propyl, butyryl, vinyl, methacryl, cinnamoyl, pivaloyl and cyclohexyl. When esters of alkenoic acids are used, the double bonds can also be utilised for polymerisation in order to build up network structures. This option also exists when using esters of dicarboxylic acids comprising the R2 radical, such as vinyl adipate, wherein crosslinking can be effected uniformly in the special polysaccharide.
Esterification reagents which are particularly suitable include vinyl acetate, vinyl propionate, vinyl laurate, vinyl butanoate, vinyl stearate, vinyl benzoate, vinyl acrylate, vinyl methacrylate, vinyl crotonate, vinyl pivalate and divinyl pivalate.
Other esterification reagents include carboxylic anhydrides, preferably acetic anhy-dride, propionic anhydride, succinic anhydride and itaconic anhydride, as well as reactive lactones, preferably propiolactone and a-angelicalactone. In the case of carboxylic anhydrides, substitution also occurs at the C6 position of the AGU as well as at the C2 position.
N-isopropylidene compounds of general formula 
can also be used as esterification reagents, whereupon the corresponding carboxylic acid esters of the oligo- and polysaccharides are formed. R1 is preferably an alkyl group comprising 2-18 C atoms, which can be saturated, unsaturated, straight-chain, branched or cyclic and which is optionally substituted, or can be an aryl group (which is optionally substituted). R1 is most preferably selected from the group comprising acetyl, propyl, butyryl, vinyl, methacryl, cinnarnoyl, pivaloyl and cyclohexyl.
Particularly suitable esterification reagents from the group comprising N-isopropylidene compounds include N-isopropylidene-O-methyl carbonate, N-isopropylidene-O-ethyl carbonate and N-isopropylidene-O-benzyl carbonate.
The method according to the invention is characterised in that the substrate is dissolved in a polar organic solventxe2x80x94which is DMSO in the case of starchxe2x80x94to which the enzyme and the transesterification reagent are added, and is subsequently incubated. The incubation temperature is between 20xc2x0 C. and 85xc2x0 C., and is preferably within the range from 20 to 45xc2x0 C., particularly in the interval from 35 to 45xc2x0 C. The times of reaction range from 2 to 100 hours, whereupon a conversion of about 50% is achieved with respect to the acylating reagent. Alternatively, the transesterification reagent can be activated by the enzyme in a preceding step. After the reaction is complete, the enzyme is separated by liquid-solid separation (e.g. centrifugation, filtration). The product is isolated by precipitation and is washed and dried. The solvents which remain can be worked-up by distillation and can subsequently be recycled to the esterification process. The enzyme can thus be used in a cyclic process without loss of activity.
When the reaction in the aforementioned system is conducted at a temperature of 40xc2x0 C., chemical esterification also occurs in addition to enzyme-catalysed esterification. This chemical esterification occurs with the production of degrees of substitution ranging from a few percent up to a maximum total degree of substitution corresponding to AS=0.25. This results in the esterification of other hydroxyl groups which are present in the molecule. This chemical esterification can be substantially suppressed if the reaction is conducted at lower temperatures (20-25xc2x0 C.), or is preferably suppressed by conducting the reaction in systems which are almost anhydrous (water content  less than 0.01%).
A partial degree of substitution of up to AS=1.0 at the C2 position of the AGU can be achieved in oligo- and polysaccharides by the method according to the invention. Any desired partial ASxe2x89xa61.0 at the C2 position of the AGU can be achieved via the molar equivalents of acylating reagent which are used (Table 1).
The partial AS in the C2 position of the AGU can also be adjusted via the reaction kinetics as well as via the molar equivalents, i.e. desired AS values xe2x89xa61.0 can be achieved depending on the time at which the reaction is stopped (Table 2).
Verification of the regioselectivity of the enzymatically catalysed esterification reaction was effected on the intact oligo- or polysaccharide by one- and multi-dimensional NMR spectrometry. For this purpose, the remaining free hydroxyl groups were esterified with a suitable carboxylic anhydride, for example with propionic anhydride for saccharide acetates or with acetic anhydride for saccharide benzoates or for other saccharide acylates. These mixed esters are soluble in chloro-form and can be investigated by NMR spectrometry. After evaluating the signals of the AGU protons as hydrocarbons via 1H/1H and 1H/13C correlation, the corresponding acyl groups can be assigned to their position on the AGU with the aid of a 1H/13C multiple bond correlation which is detected using 1H (HMBC technique) (Carbohydr. Res. 224 (1992), 277-283).
In a variant of the method according to the invention, production is effected catalysed by salts only, without further addition of enzyme. Degress of substitution  less than 1.0 at the C2 position are thereby achieved.
As distinct from enzyme catalysis, regioselectivity here is controlled via the state of dissolution of the oligo- and polysaccharides in a polar organic solvent, preferably in DMSO. Interactions between the solvents and the AGU components increase the acidity of the proton of the hydroxyl group in the C2 position of the AGU (J. Am. Chem. Soc. 98 (1976), 4386). By employing a suitable salt as a catalyst, complete esterification of this position can then be effected, wherein it is possible either to employ reaction kinetics control or to control the reaction via the type and amount of catalyst (table 3). The salt is usually present in a concentration of 1-10% by weight, preferably 2-5% by weight, with respect to the starting material.
The method results in higher product yields, and the partial degree of substitution can be adjusted in a defined manner. Suitable solvents for carrying out the method include dimethylsulphoxide (DMSO), N,N-dimethylformamide (DMF) and N,N-dimethyl-acetamide (DMA).
The method can be applied to polysaccharides, preferably to starches with different amylose contents and molecular weights, to starch derivatives such as hydroxy-ethylstarch or hydroxypropylstarch, and to pullulan and pullulan derivatives and oligosaccharides such as cyclodextrin.
Suitable catalysts include salts of inorganic mineral acids, salts of carboxylic acids and carbonates of the alkali and alkaline earth metals. The preferred salts are Na2HPO4, CaHPO4, Na2CO3, MgCO3 (NH4Cl)2CO3, Na2SO2, NH4Cl, NaBr, NaCl and LiCl, as well as sodium citrate. In order to suppress esterification at other hydroxyl groups,; e.g. in the C3 or C6 position, when using salts of weak acids as catalysts, a maximum of only 10 mol % must be used with respect to the weight of oligo- or polysaceharide to be reacted, and at the same time a defined time of reaction must be adhered to.
Esters of general formula 
are preferably used as esterification reagents, wherein R2 can denote vinyl, trihalogenoethyl or alkyl for example. Examples of R1 include an alkyl group comprising 2-18 C atoms, which can be saturated, unsaturated, straight-chain, branched or cyclic (and which is optionally substituted), or an aryl group (which is optionally substituted). When esters of alkenoic acids are used, the double bonds can also be utilised for polymerisation in order to build up network structures. This option also exists when using esters of dicarboxylic acids comprising the R2 radical, such as vinyl adipate, wherein crosslinking can be effected uniformly in the special polysaccharide.
Other esterification reagents include carboxylic anhydrides, for example acetic anhydride, propionic anhydride, succinic anhydride and itaconic anhydride, as well as reactive lactones, preferably propiolactone and a-angelicalactone. In the case of carboxylic anhydrides, substitution also occurs at the C6 position of the AGU as well as at the C2 position.
N-isopropylidene compounds of general formula 
can also be used as esterification reagents, wherein the corresponding carboxylic acid esters of the oligo- and polysaccharides are formed. Examples of R1 include alkyl groups comprising 2-18 C atoms, which can be saturated, unsaturated, straight-chain, branched or cyclic (and which are optionally substituted), and aryl groups (which are optionally substituted).
The method according to the invention is characterised in that oligo- or poly saccharides are regioselectively esterified at the hydroxyl group of position C2 of the anhydroglucose unit by active esters in polar organic solventsxe2x80x94preferably DMSOxe2x80x94 using salts as catalysts. The reaction temperature is between 20xc2x0 C. and 100xc2x0 C., and is preferably within the range from 30 to 50xc2x0 C. The times of reaction range from 0.5 to 100 hours, depending on the reaction temperature and on the catalyst used. The catalyst is separated by liquid-solid separation (e.g. centrifugation, filtration). Alternatively, a defined amount of water can also be added to the precipitant, in order to dissolve out the catalyst. The resulting ester is isolated by precipitation and is washed and dried.
The solvents which remain can be worked-up by distillation and can subsequently be recycled to the esterification process.
With the method according to the invention, a partial degree of substitution at the C2 position of the AGU of up to AS=1.0 can be achieved according to choice via the reaction kinetics or via the molar equivalents of transesterification reagent used.
Regioselectivity was verified by means of two-dimensional NMR spectrometry. For this purpose, the acylated polysaccharide had to be completely propionylated in the case of acetates, or acetylated in the case of other polysaccharide esters. In this manner, the substitution site can be unambiguously verified by means of multiple bond correlation (Carbohydr. Res. 224 (1992), 277-283).
The products which are obtained by the method according to the invention, e.g. starch acetates, can be decomposed by amylases. At a suitable molecular weight of the starch and when substitution is effected strictly at the C2 position, starch acetates are suitable as blood plasma expanders. 2-O-acetylstarch is particularly suitable for this application. Therefore, the present invention further relates to the use of 2-O-acetylstarch as a blood plasma expander. Moreover, biodegradable plastics can be synthesised from starch acylates. Membranes having a substantially uniform structure can be synthesised by crosslinking processes. Absorbents for different applications can also be produced by treatment to form further derivatives. Starch acetates which have thermoplastic properties can conceivably be used in the pharmaceutical industry as active ingredient retardants.
Regioselectively substituted cyclodextrin esters can be used in the pharmaceutical industry as carriers for pharmaceutical active ingredients. Furthermore, compounds of high molecular weight can be synthesised, in the manner of copolymers, which could be suitable for chromatography (e.g. for the separation of enantiomers).